Aerobic and anaerobic ammonia oxidizing bacteria – competitors or natural partners?

Abstract

1 Introduction

Nitrification is an important part of the biological nitrogen cycle. Microorganisms involved in nitrification are characterized as lithotrophic ammonia and nitrite oxidizing bacteria and heterotrophic nitrifiers (not discussed in this review). Lithotrophic nitrifiers are all placed in the family Nitrobacteraceae [1], although they are not necessarily related phylogenetically. Chemolithoautotrophic nitrifying bacteria have been found in many ecosystems such as fresh water, salt water, sewage systems, soils, and on/in rocks as well as in masonry [2,3 Growth under suboptimal conditions might be possible by ureolytic activity, aggregate formation [4], or in biofilms on the surfaces of substrata [5]. Nitrifiers can be found in extreme habitats at high temperatures [6] and in Antarctic soils [7,8]. Although the pH optimum for cell growth is pH 7.6–7.8, they were frequently detected in environments with pH values of about 4 such as acid tea and forest soils [9,10] and pH values of about 10 such as soda lakes [11,12] It is interesting to note that aerobic nitrifiers were also found in anoxic environments [13,14]. This is in good agreement with recent studies that show that these microorganisms have a more versatile metabolism than previously assumed. Ammonia oxidizers can denitrify with ammonia as electron donor under oxygen limited conditions [15,16] or with hydrogen or organic compounds under anoxic conditions [17]. Finally they can use N₂O₄ as oxidant for ammonia oxidation under both oxic and anoxic conditions [18]. Furthermore, a new group of anaerobic nitrite dependent ammonia oxidizers (anammox) were discovered [19,20]. This review will discuss the recent findings and their ecological importance for the understanding of the biological nitrogen cycle.

2 Anaerobic ammonium oxidation (anammox)

2.1 Molecular identity

Although Broda [21] predicted the existence of chemolithoautotrophic bacteria capable of anaerobic ammonium oxidation and Abeliovich [14] reported high cell concentrations of nitrifiers under anoxic conditions, the first experimental confirmation of anaerobic ammonia oxidation (anammox) was obtained in the early 1990s [19]. During experiments on a denitrifying pilot plant it was noted that ammonia and nitrate disappeared from the reactor effluent with a concomitant increase of dinitrogen gas production. The microbial nature of the process was verified, and nitrite was shown to be the preferred electron acceptor [22]. Hydroxylamine and hydrazine were identified as important intermediates. Since the growth rate of the anammox biomass appeared to be very low (doubling time about 11 days), reactor systems with very efficient biomass retention were necessary for the enrichment. A sequencing batch reactor system was chosen for the ecophysiological study of the anammox community [23]. The biomass in the community was dominated for more than 70% by a morphologically conspicuous bacterium. Attempts to isolate the microorganisms with ‘classical’ methods failed. Therefore, the bacterium was physically purified from enrichment cultures by density gradient centrifugation [24]. DNA extracted from the purified cells was used as a template for PCR amplification with a universal 16S rDNA primer set. The dominant 16S rDNA sequence obtained was planctomycete like, and branching very deep within the planctomycete lineage of descent (Fig. 1). The anaerobic ammonium oxidizing planctomycete like bacterium was named ‘Candidatus Brocadia anammoxidans’. The 16S rDNA sequence information was used to design specific oligonucleotide probes for application in fluorescence in situ hybridization (FISH) and to survey the presence of B. anammoxidans and related anammox bacteria in several wastewater treatment systems [25]. Indeed, B. anammoxidans and the closely related ‘Candidatus Kuenenia stuttgartiensis’ could be detected in many of these systems throughout the world and seem to be dominating in these microbial biofilm communities [25].

2.2 Molecular diversity

The order Planctomycetales, first described in 1986 by Schlessner and Stackebrandt [26], so far includes only four genera (Planctomyces, Pirellula, Gemmata, and Isosphaera) with seven validly described species [27]. Various environmentally derived 16S rDNA sequences [20,28] strongly indicate further planctomycete lineages [29], including the anammox bacteria (Fig. 1). In fact, the newly found bacterium K. stuttgartiensis forms a distinct branch within anammox bacteria and the sequence similarity of less than 90% to B. anammoxidans is indicative of a genus level diversity of these bacteria [25]. The application of FISH probes showed the dominance of these bacteria in ecosystems with high nitrogen losses. Molecular techniques are important tools to monitor the presence and activity of microorganisms in ecosystems. For example the growth rate of many bacteria can be deduced from their ribosome content [30]. This method is, however, not applicable for slow growing anammox and Nitrosomonas like bacteria [31] since inactive cells of both groups tend to keep their ribosome content at a high level. In such cases, the cellular concentrations of precursor rRNA might be a good indicator of physiological activity [32]. Therefore, the intergenic spacer regions (ISR) between the 16S rRNA and 23S rRNA, as part of the precursor rRNA, of B. anammoxidans and K. stuttgartiensis were sequenced. Subsequently, ISR targeted oligonucleotide probes were constructed and applied by FISH. Inhibition experiments with B. anammoxidans revealed a good correlation between the metabolic activity and the ISR concentrations, demonstrating the ISR targeting FISH to be a powerful method for the detection of activity changes in slow growing bacteria [31].

2.3 Ecophysiology

The ultrastructure of B. anammoxidans has many features in common with previously described planctomycetes. These microorganisms have a proteinaceous cell wall lacking peptidoglycan and are thus insensitive to ampicillin. The chromosome is separated from the surrounding cytoplasm by a single or double membrane. In B. anammoxidans an additional compartment bounded by a single membrane [33], free from ribosomes and chromosome, was observed. This peculiar ‘organelle’ made up more than 30% of the cell volume and it may play an important role in the catabolism. Using the immunogold labeling technique with antibodies against the key enzyme hydroxylamine (hydrazine) oxidoreductase [34], the enzyme was localized in this middle compartment, which was named ‘anammoxosome’[33]. Interestingly, B. anammoxidans[33] as well as aerobic ammonia oxidizers such as Nitrosomonas[1] develop internal membrane systems. Whether such a membrane system is bioenergetically necessary for ammonia oxidation is still the subject of investigation since both key enzymes of Nitrosomonas are obviously not localized in the intracytoplasmic membrane (ICM) system. According to the peptide structure the AMO was described as a membrane bound enzyme [35], and recent studies [36] indicated a localization in the cytoplasmic membrane. The HAO is localized in the periplasm [37].

To unravel the metabolic pathway for anaerobic ammonium oxidation in B. anammoxidans, series of ¹⁵N labeling experiments were conducted. It could be shown that ammonium and nitrite are combined to yield dinitrogen gas [38] and radioactive bicarbonate is incorporated in the biomass. With an excess of hydroxylamine, a transient accumulation of hydrazine was observed, indicating that hydrazine is an intermediate of the anammox process. According to the working hypothesis, the oxidation of hydrazine to dinitrogen gas is supposed to generate four electrons for the initial reduction of nitrite to hydroxylamine (Fig. 2). The overall nitrogen balance shows a ratio of about 1:1.32:0.26 for the conversion of ammonia, nitrite, and nitrate Eq. 1 The function of the formation of nitrate is assumed to be the generation of reducing equivalents necessary for the reduction of CO₂.

A high anammox activity is detectable in a pH range between 6.4 and 8.3 and a temperature range between 20 and 43°C [39]. Under optimal conditions, the specific activity is about 3.6 mmol (g protein)⁻¹ h⁻¹, the biomass yield about 0.066 C mol (mol ammonium)⁻¹, and the specific growth rate about 0.0027 h⁻¹. Recent studies showed that K. stuttgartiensis is in many ways similar to B. anammoxidans[40]. K. stuttgartiensis cells have the same overall cell structure and also produce hydrazine from exogenously supplied hydroxylamine. Energetically favorable mechanisms with Fe³⁺, Mn⁴⁺, or even sulfate as oxidant have not been reported yet [41].

To assess the occurrence of the anammox reaction in natural environments and man made ecosystems, further data about the effect of several chemical and physical parameters are necessary. For example the anammox bacteria are very sensitive to oxygen and nitrite. Oxygen concentrations as low as 2 μM and nitrite concentrations between 5 and 10 mM inhibit the anammox activity completely, but reversibly [22].

3 Ecology of anammox

In various ecosystems B. anammoxidans will be dependent on the activity of aerobic ammonia oxidizing bacteria under oxygen limited conditions, e.g., at the oxic/anoxic interface. Anammox biomass has already been detected in wastewater treatment plants in The Netherlands, Germany, Switzerland, UK, Australia, and Japan [42]. Recently anammox cells were detected in a non artificial ecosystem, a fresh water swamp in Uganda [42]. Oxic/anoxic interfaces are abundant in nature, for example in biofilms and flocs. In these oxygen limited environments the ammonia oxidizers would oxidize ammonium to nitrite and keep the oxygen concentration low, while B. anammoxidans would convert the produced nitrite and the remaining ammonium to dinitrogen gas. Such conditions have been established in many different reactor systems [16,43 43–45 [44,45]>. FISH analysis and activity measurements showed that aerobic as well as anaerobic ammonia oxidizers were present and active in these oxygen limited reactors, but aerobic nitrite oxidizers (Nitrobacter or Nitrospira) were not detected. Apparently, the aerobic nitrite oxidizers are unable to compete for oxygen with the aerobic ammonia oxidizers and for nitrite with the anaerobic ammonia oxidizers as has been documented before [46,47]. It seems likely that under these conditions anaerobic and aerobic ammonia oxidizers form a quite stable community. The cooperation of aerobic and anaerobic ammonium oxidizing bacteria is not only relevant for wastewater treatment [45,48], but might play an important role in natural environments at the oxic/anoxic interface. Further interactions under anoxic conditions between both groups of ammonia oxidizers seem to be likely since an anoxic, NO₂ dependent metabolism of Nitrosomonas like microorganisms was recently discovered [18].

4 Aerobic and anaerobic NO₂ dependent ammonia oxidation by Nitrosomonas (NO_x cycle)

4.1 Diversity

Gram negative ammonia oxidizers, e.g., members of the genera Nitrosomonas and Nitrosospira[1], are lithoautotrophic organisms using carbon dioxide as the main carbon source. Several species reveal extensive ICM systems. Recently, molecular tools to detect the presence of ammonia oxidizing bacteria in the environment have been supplemented by PCR primers for specific amplification of the ammonia monooxygenase structural gene amoA[3]. Environmental 16S rRNA and amoA libraries have extended the knowledge on the natural diversity of ammonia oxidizing bacteria [49]. Comparative 16S rRNA sequence analyses revealed that members of this physiological group are confined to two monophyletic lineages within the Proteobacteria. Nitrosococcus oceanus is affiliated with the γ subclass of the Proteobacteria, while members of the genera Nitrosomonas and Nitrosospira form a closely related group within the β subclass of Proteobacteria [50]. Using these molecular tools nitrifiers can be detected even in anoxic habitats.

4.2 Anaerobic ammonia oxidation

There are only a few differences between the anaerobic, NO₂ dependent and the aerobic, O₂ dependent [53] ammonia oxidation by Nitrosomonas. Instead of O₂ in the course of aerobic ammonia oxidation, N₂O₄ is used as electron acceptor and NO, an additional product, is released in the anaerobic ammonia oxidation. NO₂ is not available in natural environments under anoxic conditions. An anaerobic ammonia oxidation is therefore dependent on the transport of NO₂ from oxic layers.

Another important observation is that anaerobic ammonia oxidation with NO₂ (N₂O₄) as oxidant was not affected by acetylene [18]. N. eutropha cells treated with acetylene oxidized ammonia even under oxic conditions if NO₂ was available. Ammonia oxidation was not detectable in the absence of NO₂. One of the most significant findings is that the 27 kDa polypeptide of the AMO was not labeled with [¹⁴C]acetylene during anoxic NO₂ dependent ammonia oxidation. When oxygen was added, the labeling of this polypeptide with [¹⁴C]acetylene started immediately. An influence of the ammonia concentration on the labeling reaction was not observed. These studies clearly demonstrate the necessity to distinguish between NO₂ dependent and O₂ dependent ammonia oxidation. The new hypothetical model of ammonia oxidation [18] including the role of nitrogen oxides is shown in Fig. 3. Anaerobic ammonia oxidation is dependent on the presence of the oxidizing agent N₂O₄. NO is produced in stoichiometric amounts (Fig. 3). Only when NO₂ is available under anoxic conditions, ammonia is oxidized and hydroxylamine occurs as an intermediate while NO is formed as an end product. Hydroxylamine is further oxidized to nitrite [52].

Under anoxic conditions nitrite serves as a terminal electron acceptor. In the absence of ammonia Nitrosomonas is capable of using different substrates as electron donor. During hydroxylamine oxidation by ammonia oxidizers, small amounts of nitric and nitrous oxide are released [54]. Both gases are also produced in the course of aerobic denitrification by ammonia oxidizing bacteria [55,56 Additionally, the formation of dinitrogen was observed [17,57]. Furthermore, Nitrosomonas is capable of anoxic denitrification with molecular hydrogen [17] or simple organic compounds [14] serving as electron donors.

4.3 Aerobic ammonia oxidation

NO_x also plays an important role in the aerobic metabolism of nitrifying microorganisms. Nitrosomonas like organisms were distinctly inhibited when gaseous nitric oxide was removed from laboratory scale cultures by means of intensive aeration. Nitrification in these cultures only started again when nitric oxide was added to the gas inlet of the culture vessels [18,58]. The lag phase during the recovery of ammonia oxidation in starved cells could be significantly reduced when NO_x was added. Evidence is given that the cells generate NO for the NO_x cycle via denitrification when external NO_x is not available [59]. Nitrogenous oxides have a significant promoting effect on pure cultures of N. eutropha [59,60]. Their addition resulted in a pronounced increase in nitrification rate, specific activity of ammonia oxidation, growth rate, maximum cell density, and aerobic denitrification capacity. Maximum cell numbers amounted to 2×10¹⁰Nitrosomonas cells ml⁻¹. Furthermore, about 50% of the nitrite produced was aerobically denitrified to dinitrogen when nitrogen oxides were present.

The sum of Eqs. 6 and Eqs. 7, given in Eq. 8,was already described earlier as the reaction of aerobic ammonia oxidation [53], but is in complete agreement with the new hypothetical model. The total consumption rates (ammonia, oxygen) and production rates (hydroxylamine as intermediate) are the same, but the mechanism of the reaction is quite different. Since detectable NO_x concentrations were small, nitrogen oxides seem to cycle in the cell (possibly enzyme bound). Therefore, the total amount of NO_x per cells is expected to be low. This hypothetical model (Fig. 4) is in good accordance with the described mechanisms of the aerobic ammonia oxidation. According to the new model, O₂ is used to oxidize NO. The product NO₂ is then consumed during ammonia oxidation. The oxygen of hydroxylamine still originates from molecular oxygen, but is incorporated via NO₂[18].

In control experiments different species of ammonia oxidizers were tested (e.g., Nitrosomonas europaea, Nitrosolobus multiformis) [61]. All species were able to oxidize ammonia under anoxic conditions with NO₂ as oxidant (Fig. 3) and the aerobic ammonia oxidation activity was increased in the presence of NO or NO₂ (Fig. 4).

4.4 Ecological evidence of NO_x

The ecological evidence of nitrogen oxides (NO_x) for nitrification is still object of speculations and there is no simple, uniform picture of the function of NO_x in the ammonia oxidation. Further investigations are necessary to reveal the role of nitrogen oxides. First, NO₂ has to be confirmed as the master regulating signal for the ammonia oxidation [59]. The recovery of ammonia oxidation activity by denitrifying Nitrosomonas cells (hydrogen as electron donor) is regulated via the availability of NO₂. Second, in contrast to homoserine lactones, which function as signal molecules between many bacteria [62], nitrogen oxides seem to function as very specific signal molecules between ammonia oxidizers [59].

5 Conclusion

Several new microbial pathways in the nitrogen cycle have been discovered. The planctomycete like anammox bacteria converting ammonia and nitrite under anoxic conditions and the information about the flexibility of the metabolism of ‘aerobic’ nitrifiers add new possibilities to the nitrogen cycle. These two groups might even be natural partners in ecosystems with limited oxygen supply. Under these conditions aerobic ammonia oxidizers are able to oxidize ammonia to nitrite which will be consumed by anammox bacteria together with ammonia. As products of this cooperation mainly N₂ and small amounts of nitrate are detectable [43]. When ammonia is the limiting substrate the affinities of both groups of ammonia oxidizers might be decisive for the outcome of the competition. However, we are far from understanding the complexity of nitrogen conversion in detail. To gain deeper insight future studies might focus on regulation of nitrifier metabolism, on community interactions, and on phylogenetic diversity of nitrogen converting microorganisms.

References